Lettuce varieties svld9011, svld9012, svld9013, and svld9014

ABSTRACT

The invention provides seed and plants of the lettuce lines SVLD9011, SVLD9012, SVLD9013, and SVLD9014. The invention thus relates to the plants, seeds, and tissue cultures of lettuce lines SVLD9011, SVLD9012, SVLD9013, and SVLD9014, and to methods for producing a lettuce plant produced by crossing a plant of lettuce line SVLD9011, lettuce line SVLD9012, lettuce line SVLD9013, or lettuce line SVLD9014 with itself or with another lettuce plant, such as a plant of another genotype. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of a plant of lettuce lines SVLD9011, SVLD9012, SVLD9013, and SVLD9014, including the gametes of such plants.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and, morespecifically, to the development of lettuce lines SVLD9011, SVLD9012,SVLD9013, and SVLD9014.

BACKGROUND OF THE INVENTION

The goal of vegetable breeding is to combine various desirable traits ina single variety/hybrid. Such desirable traits may include any traitdeemed beneficial by a grower and/or consumer including greater yield,resistance to insects or pathogens, tolerance to environmental stress,better agronomic quality, higher nutritional value, growth rate andfruit properties.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all genetic loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different genotypes produces auniform population of hybrid plants that are heterozygous for manygenetic loci. Conversely, a cross of two plants each heterozygous at anumber of loci produces a population of hybrid plants that differgenetically and are not uniform. The resulting non-uniformity makesperformance unpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines are developed byselfing and selection of desired phenotypes. The new lines are evaluatedto determine which of those have commercial potential.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a lettuce plant of theline SVLD9011, SVLD9012, SVLD9013, or SVLD9014. Also provided arelettuce plants having all the physiological and morphologicalcharacteristics of lettuce line SVLD9011, SVLD9012, SVLD9013, orSVLD9014. Parts of the lettuce plant of the present invention are alsoprovided, for example, including pollen, an ovule, an embryo, a seed,and a cell of the plant.

The invention also concerns seed of lettuce line SVLD9011, SVLD9012,SVLD9013, or SVLD9014. The lettuce seed of the invention may be providedas an essentially homogeneous population of lettuce seed of the linedesignated SVLD9011, SVLD9012, SVLD9013, or SVLD9014. Essentiallyhomogeneous populations of seed are generally free from substantialnumbers of other seed. Therefore, in one embodiment, seed of lineSVLD9011, SVLD9012, SVLD9013, or SVLD9014 may be defined as forming atleast about 97% of the total seed, including at least about 98%, 99%, ormore of the seed. The population of lettuce seed may be particularlydefined as being essentially free from hybrid seed. The seed populationmay be separately grown to provide an essentially homogeneous populationof lettuce plants designated SVLD9011, SVLD9012, SVLD9013, or SVLD9014.

In another aspect of the invention, a plant of lettuce line SVLD9011,SVLD9012, SVLD9013, or SVLD9014 comprising an added heritable trait isprovided. The heritable trait may comprise a genetic locus that is, forexample, a dominant or recessive allele. In one embodiment of theinvention, a plant of lettuce line SVLD9011, SVLD9012, SVLD9013, orSVLD9014 is defined as comprising a single locus conversion. In specificembodiments of the invention, an added genetic locus confers one or moretraits such as, for example, herbicide tolerance, insect resistance,disease resistance, and modified carbohydrate metabolism. In furtherembodiments, the trait may be, for example, conferred by a naturallyoccurring gene introduced into the genome of the line by backcrossing, anatural or induced mutation, or a transgene introduced through genetictransformation techniques into the plant or a progenitor of any previousgeneration thereof. When introduced through transformation, a geneticlocus may comprise one or more genes integrated at a single chromosomallocation.

In some embodiments, a single locus conversion includes one or moresite-specific changes to the plant genome, such as, without limitation,one or more nucleotide modifications, deletions, or insertions. A singlelocus may comprise one or more genes or nucleotides integrated ormutated at a single chromosomal location. In one embodiment, a singlelocus conversion may be introduced by a genetic engineering technique,methods of which include, for example, genome editing with engineerednucleases (GEEN). Engineered nucleases include, but are not limited to,Cas endonucleases; zinc finger nucleases (ZFNs); transcriptionactivator-like effector nucleases (TALENs); engineered meganucleases,also known as homing endonucleases; and other endonucleases for DNA orRNA-guided genome editing that are well-known to the skilled artisan.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of line SVLD9011, SVLD9012, SVLD9013, or SVLD9014 isprovided. The tissue culture will preferably be capable of regeneratingplants capable of expressing all of the physiological and morphologicalcharacteristics of the starting plant, and of regenerating plants havingsubstantially the same genotype as the starting plant. Examples of someof the physiological and morphological characteristics of the lineSVLD9011, SVLD9012, SVLD9013, or SVLD9014 include those traits set forthin the tables herein. The regenerable cells in such tissue cultures maybe derived, for example, from embryos, meristems, cotyledons, pollen,leaves, anthers, roots, root tips, pistil, flower, seed and stalks.Still further, the present invention provides lettuce plants regeneratedfrom a tissue culture of the invention, the plants having all thephysiological and morphological characteristics of line SVLD9011,SVLD9012, SVLD9013, or SVLD9014.

In yet another aspect of the invention, processes are provided forproducing lettuce seeds and plants, which processes generally comprisecrossing a first parent lettuce plant with a second parent lettuceplant, wherein at least one of the first or second parent lettuce plantsis a plant of the line designated SVLD9011, SVLD9012, SVLD9013, orSVLD9014. These processes may be further exemplified as processes forpreparing hybrid lettuce seed or plants, wherein a first lettuce plantis crossed with a second lettuce plant of a different, distinct genotypeto provide a hybrid that has, as one of its parents, the lettuce plantof line SVLD9011, SVLD9012, SVLD9013, or SVLD9014. In these processes,crossing will result in the production of seed. The seed productionoccurs regardless of whether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent lettuce plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent lettuce plants into plants that bear flowers. A third stepmay comprise preventing self-pollination of the plants, such as byemasculating the male portions of flowers, (i.e., treating ormanipulating the flowers to produce an emasculated parent lettuceplant). Self-incompatibility systems may also be used in some hybridcrops for the same purpose. Self-incompatible plants still shed viablepollen and can pollinate plants of other varieties but are incapable ofpollinating themselves or other plants of the same line.

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent lettuce plants. Yet another step comprisesharvesting the seeds from at least one of the parent lettuce plants. Theharvested seed can be grown to produce a lettuce plant or hybrid lettuceplant.

The present invention also provides the lettuce seeds and plantsproduced by a process that comprises crossing a first parent lettuceplant with a second parent lettuce plant, wherein at least one of thefirst or second parent lettuce plants is a plant of the line designatedSVLD9011, SVLD9012, SVLD9013, or SVLD9014. In one embodiment of theinvention, lettuce seed and plants produced by the process are firstgeneration (F₁) hybrid lettuce seed and plants produced by crossing aplant in accordance with the invention with another, distinct plant. Thepresent invention further contemplates plant parts of such an F₁ hybridlettuce plant, and methods of use thereof. Therefore, certain exemplaryembodiments of the invention provide an F₁ hybrid lettuce plant and seedthereof.

In still yet another aspect of the invention, the genetic complement ofthe lettuce plant line designated SVLD9011, SVLD9012, SVLD9013, orSVLD9014 is provided. The phrase “genetic complement” is used to referto the aggregate of nucleotide sequences, the expression of whichsequences defines the phenotype of, in the present case, a lettuceplant, or a cell or tissue of that plant. A genetic complement thusrepresents the genetic makeup of a cell, tissue or plant, and a hybridgenetic complement represents the genetic make-up of a hybrid cell,tissue or plant. The invention thus provides lettuce plant cells thathave a genetic complement in accordance with the lettuce plant cellsdisclosed herein, and plants, seeds and plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that line SVLD9011, SVLD9012, SVLD9013, or SVLD9014 ora first generation progeny thereof could be identified by any of themany well-known techniques such as, for example, Simple Sequence LengthPolymorphisms (SSLPs) (Williams et al., Nucleic Acids Res., 18:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by lettuce plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a lettuce plant of the invention with a haploid geneticcomplement of a second lettuce plant, preferably, another, distinctlettuce plant. In another aspect, the present invention provides alettuce plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of lettuce line SVLD9011, SVLD9012,SVLD9013, or SVLD9014 comprising detecting in the genome of the plant atleast a first polymorphism. The method may, in certain embodiments,comprise detecting a plurality of polymorphisms in the genome of theplant. The method may further comprise storing the results of the stepof detecting the plurality of polymorphisms on a computer readablemedium. The invention further provides a computer readable mediumproduced by such a method.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from line SVLD9011, SVLD9012, SVLD9013, orSVLD9014, the method comprising the steps of: (a) preparing a progenyplant derived from line SVLD9011, SVLD9012, SVLD9013, or SVLD9014,wherein said preparing comprises crossing a plant of the line SVLD9011,SVLD9012, SVLD9013, or SVLD9014 with a second plant; and (b) crossingthe progeny plant with itself or a second plant to produce a seed of aprogeny plant of a subsequent generation. In further embodiments, themethod may additionally comprise: (c) growing a progeny plant of asubsequent generation from said seed of a progeny plant of a subsequentgeneration and crossing the progeny plant of a subsequent generationwith itself or a second plant; and repeating the steps for an additional3-10 generations to produce a plant derived from line SVLD9011,SVLD9012, SVLD9013, or SVLD9014. The plant derived from line SVLD9011,SVLD9012, SVLD9013, or SVLD9014 may be an inbred line, and theaforementioned repeated crossing steps may be defined as comprisingsufficient inbreeding to produce the inbred line. In the method, it maybe desirable to select particular plants resulting from step (c) forcontinued crossing according to steps (b) and (c). By selecting plantshaving one or more desirable traits, a plant derived from line SVLD9011,SVLD9012, SVLD9013, or SVLD9014 is obtained which possesses some of thedesirable traits of the line as well as potentially other selectedtraits.

In certain embodiments, the present invention provides a method ofproducing food comprising: (a) obtaining a plant of lettuce lineSVLD9011, SVLD9012, SVLD9013, or SVLD9014, wherein the plant has beencultivated to maturity, and (b) collecting leaf tissue from the plant,wherein the leaf tissue is capable of use as food.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

The term “about” is used to indicate that a value includes the standarddeviation of the mean for the device or method being employed todetermine the value. The use of the term “or” in the claims is used tomean “and/or” unless explicitly indicated to refer to alternatives onlyor the alternatives are mutually exclusive. When used in conjunctionwith the word “comprising” or other open language in the claims, thewords “a” and “an” denote “one or more,” unless specifically notedotherwise. The terms “comprise,” “have” and “include” are open-endedlinking verbs. Any forms or tenses of one or more of these verbs, suchas “comprises,” “comprising,” “has,” “having,” “includes” and“including,” are also open-ended. For example, any method that“comprises,” “has” or “includes” one or more steps is not limited topossessing only those one or more steps and also covers other unlistedsteps. Similarly, any plant that “comprises,” “has” or “includes” one ormore traits is not limited to possessing only those one or more traitsand covers other unlisted traits.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of the lettuce lines designated SVLD9011,SVLD9012, SVLD9013, and SVLD9014. These lines show uniformity andstability within the limits of environmental influence for the traitsdescribed hereinafter. Lettuce lines SVLD9011, SVLD9012, SVLD9013, andSVLD9014 provide sufficient seed yield. By crossing with a distinctsecond plant, uniform F₁ hybrid progeny can be obtained.

Lettuce line SVLD9011, also known as 19-8W-CHD-9011, lettuce lineSVLD9012, also known as 19-8W-CHD-9012, lettuce line SVLD9013, alsoknown as 19-8W-CHD-9013, and lettuce line SVLD9014, also known as19-8W-CHD-9014, are iceberg lettuce varieties.

A. Physiological and Morphological Characteristics of Lettuce LinesSVLD9011, SVLD9012, SVLD9013, and SVLD9014

In accordance with one aspect of the present invention, there isprovided a plant having the physiological and morphologicalcharacteristics of lettuce line SVLD9011, SVLD9012, SVLD9013, orSVLD9014. A description of the physiological and morphologicalcharacteristics of lettuce lines SVLD9011, SVLD9012, SVLD9013, andSVLD9014 are presented in the tables below.

TABLE 1 Physiological and Morphological Characteristics of Lettuce LineSVLD9011 CHARACTERISTIC SVLD9011 Vanguard Denver Plant type vanguardgroup vanguard group vanguard group spread of frame leaves (cm) 54.053.6 48.6 diameter very large very large very large height (floweringplant) very tall very tall very tall fasciation (at flowering absentabsent absent stage) head formation closed head closed head closed head(overlapping) (overlapping) (overlapping) head, degree of overlappingvery strong very strong very strong of upper part of plant head diameter(cm) 20.0 18.4 18.0 head shape spherical spherical spherical head shapein longitudinal circular circular circular section head size (class)large large large head weight (gms) 1197.04 960.4 1129.3 headfirmness/density firm/dense firm/dense firm/dense core, diameter at thebase of 35.3 39.8 34.5 the head (mm) core, ratio of head diameter/ 5.74.6 5.2 core diameter (decimals) core, core height from the 84.9 47.664.1 base of the head to the apex (mm) axillary sprouting strong strongmedium time of harvest maturity late late late Bolting time of beginningof bolting late late late under long day conditions first water dateJun. 20, 2019 Jun. 20, 2019 Jun. 20, 2019 number of days from first 6766 69 water date to seed stalk emergence (summer conditions) boltingclass slow slow slow height of mature seed stalk 150.4 143.5 165.6 (cm)spread of the bolter plant at 54.3 44.95 52.00 the widest point (cm)bolter leaves curved curved curved margin dentate dentate dentate bolterhabit: terminal present present present inflorescence bolter habit:lateral shoots present present present bolter habit: basal side absentabsent absent shoots Leaf leaf blade, incisions of present presentpresent margin on apical part leaf blade, depth of incisions moderate/moderate/ moderate/ on margin on apical part medium medium medium(harvest mature outer leaves) leaf blade, density of sparse sparsesparse incisions on margin on apical part leaf blade, type of incisionssinuate sinuate sinuate on apical part leaf blade, venation flabellateflabellate flabellate leaf blade, degree of weak to moderate/ moderate/undulation of apical margin moderate/ medium medium (harvest matureouter leaves) medium indentation (finest divisions shallowly shallowlyshallowly of the margin) (harvest dentate dentate dentate mature outerleaves) green color (harvest mature medium green medium green mediumgreen outer leaves) hue of green color (harvest greyish greyish greyishmature outer leaves) intensity of color (harvest medium medium mediummature outer leaves) anthocyanin coloration absent absent absent sizelarge large large glossiness (harvest mature dull dull dull outerleaves) glossiness of upper side weak weak weak blistering (harvestmature weak to moderate/ weak outer leaves) moderate/ medium medium sizeof blisters medium medium medium thickness (harvest mature medium mediummedium outer leaves) trichomes (harvest mature absent absent absentouter leaves) (smooth) (smooth) (smooth) attitude at harvest maturityhorizontal horizontal horizontal (outer leaves from head lettuce oradult leaves from cutting and stem lettuce) shape transverse broadtransverse broad transverse broad elliptic elliptic elliptic shape oftip rounded rounded rounded shape of fourth leaf oval oval ovallength/width index of fourth 20.4 26.5 20.5 leaf apical margin(cotyledon to moderately moderately moderately 4^(th) leaf stage)dentate dentate dentate basal margin (cotyledon to entire entire entire4^(th) leaf stage) undulation (cotyledon to 4^(th) slight slight slightleaf stage) green color (cotyledon to 4^(th) dark green dark green darkgreen leaf stage) anthocyanin distribution absent absent absent(cotyledon to 4^(th) leaf stage) rolling (cotyledon to 4^(th) leafabsent absent absent stage) cupping (cotyledon to 4^(th) uncuppeduncupped uncupped leaf stage) reflexing (cotyledon to 4^(th) none nonenone leaf stage) attitude at 10-12 leaf stage semi-erect semi-erectsemi-erect division at 10-12 leaf stage entire entire entire butt, shapeflat flat flat butt, midrib moderately moderately moderately raisedraised raised Seed color black black black light dormancy light notrequired light not required light not required heat dormancy susceptiblesusceptible susceptible Seedling anthocyanin coloration absent absentabsent size of cotyledon (fully medium medium medium developed) shape ofcotyledon medium elliptic medium elliptic medium elliptic shape ofcotyledons intermediate intermediate intermediate Maturity earliness ofharvest-mature 53 53 53 head formation (number of days from first waterdate to harvest) These are typical values. Values may vary due toenvironment. Other values that are substantially equivalent are withinthe scope of the invention.

TABLE 2 Physiological and Morphological Characteristics of Lettuce LineSVLD9012 CHARACTERISTIC SVLD9012 Vanguard Denver Plant type vanguardgroup vanguard group vanguard group spread of frame leaves (cm) 49.053.6 48.6 diameter very large very large very large height (floweringplant) very tall tall tall fasciation (at flowering absent absent absentstage) head formation closed head closed head closed head (overlapping)(overlapping) (overlapping) head, degree of overlapping very strong verystrong very strong of upper part of plant head diameter (cm) 17.3 18.418.0 head shape spherical spherical spherical head shape in longitudinalcircular circular circular section head size (class) large large largehead weight (gms) 1115.9 960.4 1129.3 head firmness/density firm/densefirm/dense firm/dense core, diameter at the base of 35.2 39.8 34.5 thehead (mm) core, ratio of head diameter/ 4.9 4.6 5.2 core diameter(decimals) core, core height from the 73.3 47.6 64.1 base of the head tothe apex (mm) axillary sprouting strong strong medium time of harvestmaturity late late late Bolting time of beginning of bolting late latelate under long day conditions first water date Jun. 20, 2019 Jun. 20,2019 Jun. 20, 2019 number of days from first 67 66 69 water date to seedstalk emergence (summer conditions) bolting class slow slow slow heightof mature seed stalk 148.75 143.5 165.6 (cm) spread of the bolter plantat 50.10 45.00 52.00 the widest point (cm) bolter leaves curved curvedcurved margin dentate dentate dentate color dark green dark green darkgreen bolter habit: terminal present present present inflorescencebolter habit: lateral shoots present present present bolter habit: basalside absent absent absent shoots Leaf leaf blade, incisions of presentpresent present margin on apical part leaf blade, depth of incisionsmoderate/ moderate/ moderate/ on margin on apical part medium mediummedium (harvest mature outer leaves) leaf blade, density ofsparse/medium sparse sparse incisions on margin on apical part leafblade, type of incisions sinuate sinuate sinuate on apical part leafblade, venation flabellate flabellate flabellate leaf blade, degree ofweak to moderate/ moderate/ undulation of apical margin moderate/ mediummedium (harvest mature outer leaves) medium indentation (finestdivisions shallowly shallowly shallowly of the margin) (harvest dentatedentate dentate mature outer leaves) green color (harvest mature mediumgreen medium green medium green outer leaves) hue of green color(harvest greyish greyish greyish mature outer leaves) intensity of color(harvest medium medium medium mature outer leaves) anthocyanincoloration absent absent absent size large large large glossiness(harvest mature dull dull dull outer leaves) glossiness of upper sideweak weak weak blistering (harvest mature moderate/ moderate/ weak outerleaves) medium medium size of blisters medium medium medium thickness(harvest mature medium medium medium outer leaves) trichomes (harvestmature absent absent absent outer leaves) (smooth) (smooth) (smooth)attitude at harvest maturity horizontal horizontal horizontal (outerleaves from head lettuce or adult leaves from cutting and stem lettuce)shape transverse broad transverse broad transverse broad ellipticelliptic elliptic shape of tip rounded rounded rounded spread of frameleaves (cm) 49.0 53.6 48.6 shape of fourth leaf oval oval ovallength/width index of fourth 18.8 26.5 20.5 leaf apical margin(cotyledon to moderately moderately moderately 4^(th) leaf stage)dentate dentate dentate basal margin (cotyledon to entire entire entire4^(th) leaf stage) undulation (cotyledon to 4^(th) slight slight slightleaf stage) green color (cotyledon to 4^(th) dark green dark green darkgreen leaf stage) anthocyanin distribution absent absent absent(cotyledon to 4^(th) leaf stage) rolling (cotyledon to 4^(th) leafabsent absent absent stage) cupping (cotyledon to 4^(th) uncuppeduncupped uncupped leaf stage) reflexing (cotyledon to 4^(th) none nonenone leaf stage) attitude at 10-12 leaf stage semi-erect semi-erectsemi-erect division at 10-12 leaf stage entire entire entire butt, shapeflat flat flat butt, midrib moderately moderately moderately raisedraised raised Seed color black black black light dormancy light notrequired light not required light not required heat dormancy susceptiblesusceptible susceptible Seedling anthocyanin coloration absent absentabsent size of cotyledon (fully medium medium medium developed) shape ofcotyledon medium elliptic medium elliptic medium elliptic shape ofcotyledons intermediate intermediate intermediate Maturity earliness ofharvest-mature 53 53 53 head formation (number of days from first waterdate to harvest) These are typical values. Values may vary due toenvironment. Other values that are substantially equivalent are withinthe scope of the invention.

TABLE 3 Physiological and Morphological Characteristics of Lettuce LineSVLD9013 CHARACTERISTIC SVLD9013 Vanguard Denver Plant type vanguardgroup vanguard group vanguard group spread of frame leaves (cm) 51.653.6 48.6 diameter very large very large very large height (floweringplant) very tall tall tall fasciation (at flowering absent absent absentstage) head formation closed head closed head closed head (overlapping)(overlapping) (overlapping) head, degree of overlapping very strong verystrong very strong of upper part of plant head diameter (cm) 17.3 18.418.0 head shape spherical spherical spherical head shape in longitudinalcircular circular circular section head size (class) large large largehead weight (gms) 1017.0 960.4 1129.3 head firmness/density firm/densefirm/dense firm/dense core, diameter at the base of 35.4 39.8 34.5 thehead (mm) core, ratio of head diameter/ 4.9 4.6 5.2 core diameter(decimals) core, core height from the 69.8 47.6 64.1 base of the head tothe apex (mm) axillary sprouting very strong strong medium time ofharvest maturity late late late Bolting time of beginning of boltinglate late late under long day conditions first water date Jun. 20, 2019Jun. 20, 2019 Jun. 20, 2019 number of days from first 67 66 69 waterdate to seed stalk emergence (summer conditions) bolting class slow slowslow height of mature seed stalk 153.1 143.5 165.6 (cm) spread of thebolter plant at 52.90 45.00 52.00 the widest point (cm) bolter leavescurved curved curved margin dentate dentate dentate color dark greendark green dark green bolter habit: terminal present present presentinflorescence bolter habit: lateral shoots present present presentbolter habit: basal side absent absent absent shoots Leaf leaf blade,incisions of present present present margin on apical part leaf blade,depth of incisions moderate/ moderate/ moderate/ on margin on apicalpart medium medium medium (harvest mature outer leaves) leaf blade,density of sparse/medium sparse sparse incisions on margin on apicalpart leaf blade, type of incisions sinuate sinuate sinuate on apicalpart leaf blade, venation flabellate flabellate flabellate leaf blade,degree of moderate/ moderate/ moderate/ undulation of apical marginmedium medium medium (harvest mature outer leaves) indentation (finestdivisions shallowly shallowly shallowly of the margin) (harvest dentatedentate dentate mature outer leaves) green color (harvest mature mediumgreen medium green medium green outer leaves) hue of green color(harvest greyish greyish greyish mature outer leaves) intensity of color(harvest medium medium medium mature outer leaves) anthocyanincoloration absent absent absent size large large large glossiness(harvest mature dull dull dull outer leaves) glossiness of upper sideweak weak weak blistering (harvest mature weak to moderate/ weak outerleaves) moderate/ medium medium size of blisters medium medium mediumthickness (harvest mature medium medium medium outer leaves) trichomes(harvest mature absent absent absent outer leaves) (smooth) (smooth)(smooth) attitude at harvest maturity horizontal horizontal horizontal(outer leaves from head lettuce or adult leaves from cutting and stemlettuce) shape transverse broad transverse broad transverse broadelliptic elliptic elliptic shape of tip rounded rounded rounded spreadof frame leaves (cm) 51.6 53.6 48.6 shape of fourth leaf oval oval ovallength/width index of fourth 20.5 26.5 20.5 leaf apical margin(cotyledon to moderately moderately moderately 4^(th) leaf stage)dentate dentate dentate basal margin (cotyledon to entire entire entire4^(th) leaf stage) undulation (cotyledon to 4^(th) slight slight slightleaf stage) green color (cotyledon to 4^(th) dark green dark green darkgreen leaf stage) anthocyanin distribution absent absent absent(cotyledon to 4^(th) leaf stage) rolling (cotyledon to 4^(th) leafabsent absent absent stage) cupping (cotyledon to 4^(th) uncuppeduncupped uncupped leaf stage) reflexing (cotyledon to 4^(th) none nonenone leaf stage) attitude at 10-12 leaf stage semi-erect semi-erectsemi-erect division at 10-12 leaf stage entire entire entire butt, shapeflat flat flat butt, midrib moderately moderately moderately raisedraised raised Seed color black black black light dormancy light notrequired light not required light not required heat dormancy susceptiblesusceptible susceptible Seedling anthocyanin coloration absent absentabsent size of cotyledon (fully medium medium medium developed) shape ofcotyledon medium elliptic medium elliptic medium elliptic shape ofcotyledons intermediate intermediate intermediate Maturity earliness ofharvest-mature 53 53 53 head formation (number of days from first waterdate to harvest) These are typical values. Values may vary due toenvironment. Other values that are substantially equivalent are withinthe scope of the invention.

TABLE 4 Physiological and Morphological Characteristics of Lettuce LineSVLD9014 CHARACTERISTIC SVLD9014 Vanguard Denver Plant type vanguardgroup vanguard group vanguard group spread of frame leaves (cm) 50.853.6 48.6 diameter very large very large very large height (floweringplant) very tall tall tall fasciation (at flowering absent absent absentstage) head formation closed head closed head closed head (overlapping)(overlapping) (overlapping) head, degree of overlapping very strong verystrong very strong of upper part of plant head diameter (cm) 17.5 18.418.0 head shape spherical spherical spherical head shape in longitudinalcircular circular circular section head size (class) large large largehead weight (gms) 1141.8 960.4 1129.3 head firmness/density firm/densefirm/dense firm/dense core, diameter at the base of 33.8 39.8 34.5 thehead (mm) core, ratio of head diameter/ 5.2 4.6 5.2 core diameter(decimals) core, core height from the 83.5 47.6 64.1 base of the head tothe apex (mm) axillary sprouting strong strong medium time of harvestmaturity late late late Bolting time of beginning of bolting late latelate under long day conditions first water date Jun. 20, 2019 Jun. 20,2019 Jun. 20, 2019 number of days from first 67 66 69 water date to seedstalk emergence (summer conditions) bolting class slow slow slow heightof mature seed stalk 151.8 143.5 165.6 (cm) spread of the bolter plantat 48.30 45.00 52.00 the widest point (cm) bolter leaves straight curvedcurved margin dentate dentate dentate color medium green dark green darkgreen bolter habit: terminal present present present inflorescencebolter habit: lateral shoots present present present bolter habit: basalside absent absent absent shoots Leaf leaf blade, incisions of presentpresent present margin on apical part leaf blade, depth of incisionsmoderate/ moderate/ moderate/ on margin on apical part medium mediummedium (harvest mature outer leaves) leaf blade, density ofsparse/medium sparse sparse incisions on margin on apical part leafblade, type of incisions sinuate sinuate sinuate on apical part leafblade, venation flabellate flabellate flabellate leaf blade, degree ofmoderate/ moderate/ moderate/ undulation of apical margin medium mediummedium (harvest mature outer leaves) indentation (finest divisionsshallowly shallowly shallowly of the margin) (harvest dentate dentatedentate mature outer leaves) green color (harvest mature medium greenmedium green medium green outer leaves) hue of green color (harvestgreyish greyish greyish mature outer leaves) intensity of color (harvestmedium medium medium mature outer leaves) anthocyanin coloration absentabsent absent size large large large glossiness (harvest mature dulldull dull outer leaves) glossiness of upper side weak weak weakblistering (harvest mature moderate/ moderate/ weak outer leaves) mediummedium size of blisters medium medium medium thickness (harvest maturemedium medium medium outer leaves) trichomes (harvest mature absentabsent absent outer leaves) (smooth) (smooth) (smooth) attitude atharvest maturity horizontal horizontal horizontal (outer leaves fromhead lettuce or adult leaves from cutting and stem lettuce) shapetransverse broad transverse broad transverse broad elliptic ellipticelliptic shape of tip rounded rounded rounded spread of frame leaves(cm) 50.8 53.6 48.6 shape of fourth leaf oval oval oval length/widthindex of fourth 19.4 26.5 20.5 leaf apical margin (cotyledon tomoderately moderately moderately 4^(th) leaf stage) dentate dentatedentate basal margin (cotyledon to entire entire entire 4^(th) leafstage) undulation (cotyledon to 4^(th) slight slight slight leaf stage)green color (cotyledon to 4^(th) dark green dark green dark green leafstage) anthocyanin distribution absent absent absent (cotyledon to4^(th) leaf stage) rolling (cotyledon to 4^(th) leaf absent absentabsent stage) cupping (cotyledon to 4^(th) uncupped uncupped uncuppedleaf stage) reflexing (cotyledon to 4^(th) none none none leaf stage)attitude at 10-12 leaf stage semi-erect semi-erect semi-erect divisionat 10-12 leaf stage entire entire entire butt, shape flat flat flatbutt, midrib moderately moderately moderately raised raised raised Seedcolor black black black light dormancy light not required light notrequired light not required heat dormancy susceptible susceptiblesusceptible Seedling anthocyanin coloration absent absent absent size ofcotyledon (fully medium medium medium developed) shape of cotyledonmedium elliptic medium elliptic medium elliptic shape of cotyledonsintermediate intermediate intermediate Maturity earliness ofharvest-mature 53 53 53 head formation (number of days from first waterdate to harvest) These are typical values. Values may vary due toenvironment. Other values that are substantially equivalent are withinthe scope of the invention.

B. Breeding Lettuce Plants

One aspect of the current invention concerns methods for crossing thelettuce line SVLD9011, SVLD9012, SVLD9013, or SVLD9014 with itself or asecond plant and the seeds and plants produced by such methods. Thesemethods can be used for propagation of line SVLD9011, SVLD9012,SVLD9013, or SVLD9014, or can be used to produce hybrid lettuce seedsand the plants grown therefrom. Hybrid seeds are produced by crossingline SVLD9011, SVLD9012, SVLD9013, or SVLD9014 with second lettuceparent line.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing line SVLD9011, SVLD9012, SVLD9013, orSVLD9014 followed by multiple generations of breeding according to suchwell-known methods. New varieties may be created by crossing with anysecond plant. In selecting such a second plant to cross for the purposeof developing novel lines, it may be desired to choose those plantswhich either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) inprogeny. Once initial crosses have been made, inbreeding and selectiontake place to produce new varieties. For development of a uniform line,often five or more generations of selfing and selection are involved.

Uniform lines of new varieties may also be developed by way ofdouble-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g. colchicine treatment). Alternatively, haploid embryos may be growninto haploid plants and treated to induce chromosome doubling. In eithercase, fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with lineSVLD9011, SVLD9012, SVLD9013, or SVLD9014 and progeny thereof to achievea homozygous line.

New varieties may be created, for example, by crossing line SVLD9011,SVLD9012, SVLD9013, or SVLD9014 with any second plant and selection ofprogeny in various generations and/or by doubled haploid technology. Inchoosing a second plant to cross for the purpose of developing novellines, it may be desired to choose those plants which either themselvesexhibit one or more selected desirable characteristics or which exhibitthe desired characteristic(s) in progeny. After one or more lines arecrossed, true-breeding lines may be developed.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny have the characteristicbeing transferred, but are like the superior parent for most or almostall other loci. The last backcross generation would be selfed to givepure breeding progeny for the trait being transferred.

The line of the present invention is particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the line. In selecting a second plant to cross withSVLD9011, SVLD9012, SVLD9013, or SVLD9014 for the purpose of developingnovel lettuce lines, it will typically be preferred to choose thoseplants which either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) when inhybrid combination. Examples of desirable characteristics may include,for example, seed yield, seed size, seed shape, seed uniformity, earlymaturity, disease resistance, herbicide tolerance, seedling vigor,adaptability for soil conditions, and adaptability for climateconditions.

C. Further Embodiments of the Invention

In certain aspects of the invention, plants described herein modified toinclude at least a first desired heritable trait are provided. Suchplants may, in one embodiment, be developed by a plant breedingtechnique called backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to a genetic locus transferred into the plant viathe backcrossing technique. The term single locus converted plant asused herein refers to those lettuce plants which are developed by aplant breeding technique called backcrossing or by genetic engineering,wherein essentially all of the desired morphological and physiologicalcharacteristics of a variety are recovered or conserved in addition tothe single locus introduced into the variety via the backcrossing orgenetic engineering technique, respectively. By essentially all of themorphological and physiological characteristics, it is meant that thecharacteristics of a plant are recovered or conserved that are otherwisepresent when compared in the same environment, other than an occasionalvariant trait that might arise during backcrossing, introduction of atransgene, or application of genetic engineering. It is understood thata locus introduced by backcrossing may or may not be transgenic inorigin, and thus the term backcrossing specifically includesbackcrossing to introduce loci that were created by introduction of atransgene or application of genetic engineering.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentallettuce plant which contributes the locus for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental lettuce plant towhich the locus or loci from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a lettuce plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred locus fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered and the genetic distance between the recurrentand nonrecurrent parents. Although backcrossing methods are simplifiedwhen the characteristic being transferred is a dominant allele, arecessive allele, or an additive allele (between recessive anddominant), may also be transferred. In this instance it may be necessaryto introduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

In one embodiment, progeny lettuce plants of a backcross in whichSVLD9011, SVLD9012, SVLD9013, or SVLD9014 is the recurrent parentcomprise (i) the desired trait from the non-recurrent parent and (ii)all of the physiological and morphological characteristics of lettuceline SVLD9011, SVLD9012, SVLD9013, or SVLD9014 as determined at the 5%significance level when grown in the same environmental conditions.

Lettuce varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

With the development of molecular markers associated with particulartraits, it is possible to add additional traits into an established germline, such as represented here, with the end result being substantiallythe same base germplasm with the addition of a new trait or traits.Molecular breeding, as described in Moose and Mumm, 2008 (PlantPhysiol., 147: 969-977), for example, and elsewhere, provides amechanism for integrating single or multiple traits or QTL into an eliteline. This molecular breeding-facilitated movement of a trait or traitsinto an elite line may encompass incorporation of a particular genomicfragment associated with a particular trait of interest into the eliteline by the mechanism of identification of the integrated genomicfragment with the use of flanking or associated marker assays. In theembodiment represented here, one, two, three or four genomic loci, forexample, may be integrated into an elite line via this methodology. Whenthis elite line containing the additional loci is further crossed withanother parental elite line to produce hybrid offspring, it is possibleto then incorporate at least eight separate additional loci into thehybrid. These additional loci may confer, for example, such traits as adisease resistance or a fruit quality trait. In one embodiment, eachlocus may confer a separate trait. In another embodiment, loci may needto be homozygous and exist in each parent line to confer a trait in thehybrid. In yet another embodiment, multiple loci may be combined toconfer a single robust phenotype of a desired trait.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,modified fatty acid or carbohydrate metabolism, and enhanced nutritionalquality. These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. An example of a dominant trait is the downy mildewresistance trait. For this selection process, the progeny of the initialcross are sprayed with downy mildew spores prior to the backcrossing.The spraying eliminates any plants which do not have the desired downymildew resistance characteristic, and only those plants which have thedowny mildew resistance gene are used in the subsequent backcross. Thisprocess is then repeated for all additional backcross generations.

Selection of lettuce plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection applicable to the breeding oflettuce are well known in the art. Such methods will be of particularutility in the case of recessive traits and variable phenotypes, orwhere conventional assays may be more expensive, time consuming orotherwise disadvantageous. In addition, marker assisted selection may beused to identify plants comprising desirable genotypes at the seed,seedling, or plant stage, to identify or assess the purity of acultivar, to catalog the genetic diversity of a germplasm collection,and to monitor specific alleles or haplotypes within an establishedcultivar.

Types of genetic markers which could be used in accordance with theinvention include, but are not necessarily limited to, Simple SequenceLength Polymorphisms (SSLPs) (Williams et al., Nucleic Acids Res., 18:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In particular embodiments of the invention, marker assisted selection isused to increase the efficiency of a backcrossing breeding scheme forproducing lettuce line comprising a desired trait. This technique iscommonly referred to as marker assisted backcrossing (MABC). Thistechnique is well-known in the art and may involve, for example, the useof three or more levels of selection, including foreground selection toidentity the presence of a desired locus, which may complement orreplace phenotype screening protocols; recombinant selection to minimizelinkage drag; and background selection to maximize recurrent parentgenome recovery.

D. Plants Derived by Genetic Engineering

Various genetic engineering technologies have been developed and may beused by those of skill in the art to introduce traits in plants. Incertain aspects of the claimed invention, traits are introduced intolettuce plants via altering or introducing a single genetic locus ortransgene into the genome of a recited variety or progenitor thereof.Methods of genetic engineering to modify, delete, or insert genes andpolynucleotides into the genomic DNA of plants are well-known in theart.

In specific embodiments of the invention, improved lettuce lines can becreated through the site-specific modification of a plant genome.Methods of genetic engineering include, for example, utilizingsequence-specific nucleases such as zinc-finger nucleases (see, forexample, U.S. Pat. Appl. Pub. No. 2011-0203012); engineered or nativemeganucleases; TALE-endonucleases (see, for example, U.S. Pat. Nos.8,586,363 and 9,181,535); and RNA-guided endonucleases, such as those ofthe CRISPR/Cas systems (see, for example, U.S. Pat. Nos. 8,697,359 and8,771,945 and U.S. Pat. Appl. Pub. No. 2014-0068797). One embodiment ofthe invention thus relates to utilizing a nuclease or any associatedprotein to carry out genome modification. This nuclease could beprovided heterologously within donor template DNA for templated-genomicediting or in a separate molecule or vector. A recombinant DNA constructmay also comprise a sequence encoding one or more guide RNAs to directthe nuclease to the site within the plant genome to be modified. Furthermethods for altering or introducing a single genetic locus include, forexample, utilizing single-stranded oligonucleotides to introduce basepair modifications in a lettuce plant genome (see, for example Sauer etal., Plant Physiol, 170(4):1917-1928, 2016).

Methods for site-directed alteration or introduction of a single geneticlocus are well-known in the art and include those that utilizesequence-specific nucleases, such as the aforementioned, or complexes ofproteins and guide-RNA that cut genomic DNA to produce a double-strandbreak (DSB) or nick at a genetic locus. As is well-understood in theart, during the process of repairing the DSB or nick introduced by thenuclease enzyme, a donor template, transgene, or expression cassettepolynucleotide may become integrated into the genome at the site of theDSB or nick. The presence of homology arms in the DNA to be integratedmay promote the adoption and targeting of the insertion sequence intothe plant genome during the repair process through homologousrecombination or non-homologous end joining (NHEJ).

In another embodiment of the invention, genetic transformation may beused to insert a selected transgene into a plant of the invention ormay, alternatively, be used for the preparation of transgenes which canbe introduced by backcrossing. Methods for the transformation of plantsthat are well-known to those of skill in the art and applicable to manycrop species include, but are not limited to,

Vectors used for the transformation of lettuce cells are not limited solong as the vector can express an inserted DNA in the cells. Forexample, vectors comprising promoters for constitutive gene expressionin lettuce cells (e.g., cauliflower mosaic virus 35S promoter) andpromoters inducible by exogenous stimuli can be used. Examples ofsuitable vectors include pBI binary vector. The “lettuce cell” intowhich the vector is to be introduced includes various forms of lettucecells, such as cultured cell suspensions, protoplasts, leaf sections,and callus.

A vector can be introduced into lettuce cells by known methods, such asthe polyethylene glycol method, polycation method, electroporation,Agrobacterium-mediated transfer, particle bombardment and direct DNAuptake by protoplasts. See, e.g., Pang et al. (The Plant J., 9, 899-909,1996).

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner. An example of electroporation of lettuceprotoplasts is presented in Chupeau et al. (Nat. Biotechnol., 7:503-508,1989).

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target lettuce cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing the damage inflicted on the recipient cells by projectiles thatare too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples involvingmicroprojectile bombardment transformation with lettuce can be found in,for example, Elliott et al. (Plant Cell Rep., 18:707-714, 2004) andMolinier et al. (Plant Cell Rep., 21:251-256, 2002).

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Bio-Technology, 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Nat. Biotechnol., 3:629-635, 1985; U.S.Pat. No. 5,563,055). For example, U.S. Pat. No. 5,349,124 describes amethod of transforming lettuce plant cells using Agrobacterium-mediatedtransformation. By inserting a chimeric gene having a DNA codingsequence encoding for the full-length B.t. toxin protein that expressesa protein toxic toward Lepidopteran larvae, this methodology resulted inlettuce having resistance to such insects.

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al.,Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986;Marcotte et al., Nature, 335:454, 1988). Transformation of plants andexpression of foreign genetic elements is exemplified in Choi et al.(Plant Cell Rep., 13: 344-348, 1994) and Ellul et al. (Theor. Appl.Genet., 107:462-469, 2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for lettuce plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, which confers constitutive, high-level expressionin most plant tissues (see, e.g., Odel et al., Nature, 313:810, 1985),including monocots (see, e.g., Dekeyser et al., Plant Cell, 2:591, 1990;Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990); a tandemlyduplicated version of the CaMV 35S promoter, the enhanced 35S promoter(P-e35S) the nopaline synthase promoter (An et al., Plant Physiol.,88:547, 1988), the octopine synthase promoter (Fromm et al., Plant Cell,1:977, 1989); and the figwort mosaic virus (P-FMV) promoter as describedin U.S. Pat. No. 5,378,619 and an enhanced version of the FMV promoter(P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem,the cauliflower mosaic virus 19S promoter, a sugarcane bacilliform viruspromoter, a commelina yellow mottle virus promoter, and other plant DNAvirus promoters known to express in plant cells.

With an inducible promoter the rate of transcription increases inresponse to an inducing agent. Any inducible promoter can be used in theinstant invention. A variety of plant gene promoters that are regulatedin response to environmental, hormonal, chemical, and/or developmentalsignals can be used for expression of an operably linked gene in plantcells, including promoters regulated by (1) heat (Callis et al., PlantPhysiol., 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter,Kuhlemeier et al., Plant Cell, 1:471, 1989; maize rbcS promoter,Schaffner and Sheen, Plant Cell, 3:997, 1991; or chlorophyll a/b-bindingprotein promoter, Simpson et al., EMBO J., 4:2723, 1985), (3) hormones,such as abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4)wounding (e.g., wunl, Siebertz et al., Plant Cell, 1:961, 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters (e.g., Roshal etal., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988;Bustos et al., Plant Cell, 1:839, 1989). Exemplary organ-specific ororgan-preferred promoters include, but are not limited to, aroot-preferred promoter, such as that from the phaseolin gene(Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA, 82:3320-3324,1985); a leaf-specific and light-induced promoter such as that from cabor rubisco (Simpson et al., EMBO J., 4:2723, 1985) and Timko et al.,Nature, 318:579-582, 1985); an anther-specific promoter such as thatfrom LAT52 (Twell et al., Mol. Gen. Genetics, 217:240-245, 1989); apollen-specific promoter such as that from Zm13 (Guerrero et al., Mol.Gen. Genetics, 244:161-168, 1993) or a microspore-preferred promotersuch as that from apg (Twell et al., Sex. Plant Reprod., 6:217-224,1993).

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion or for secretion into the apoplast, may be accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Many signal sequences are known in the art. See, for example Becker etal. (Plant Mol. Biol., 20:49, 1992); Knox et al. (Plant Mol. Biol.,9:3-17, 1987); Lerner et al. (Plant Physiol., 91:124-129, 1989); Fonteset al. (Plant Cell, 3:483-496, 1991); Matsuoka et al. (Proc. Natl. Acad.Sci. USA, 88:834, 1991); Gould et al. (J. Cell. Biol., 108:1657, 1989);Creissen et al. (Plant J., 2:129, 1991); Kalderon et al. (Cell,39:499-509, 1984); Steifel et al. (Plant Cell, 2:785-793, 1990).

Exemplary nucleic acids which may be introduced to the lettuce lines ofthis invention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a lettuce plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a lettuce plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference in their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillito, Mol.Biotech., 7:125,1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

E. Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

Allele: Any of one or more alternative forms of a genetic locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration: The development of a plant from tissue culture.

Resistance: As used herein, the terms “resistance” and “tolerance” areused interchangeably to describe plants that show no symptoms to aspecified biotic pest, pathogen, abiotic influence or environmentalcondition. These terms are also used to describe plants showing somesymptoms but that are still able to produce marketable product with anacceptable yield. Some plants that are referred to as resistant ortolerant are only so in the sense that they may still produce a crop,even though the plants are stunted and the yield is reduced.

Royal Horticultural Society (RHS) Colour Chart Value: The RHS ColourChart is a standardized reference which allows accurate identificationof any color. A color's designation on the chart describes its hue,brightness and saturation. A color is precisely named by the RHS ColourChart by identifying the group name, sheet number and letter, e.g.,Yellow-Orange Group 19A or Red Group 41B.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing or genetic engineering ofa locus wherein essentially all of the morphological and physiologicalcharacteristics of a lettuce variety are recovered or conserved inaddition to the characteristics of the single locus.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tetraploid: A cell or organism having four sets of chromosomes.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a lettuce plant by transformation orsite-specific modification.

Triploid: A cell or organism having three sets of chromosomes.

F. DEPOSIT INFORMATION

A deposit of lettuce line SVLD9011, lettuce line SVLD9012, lettuce lineSVLD9013, and lettuce line SVLD9014, disclosed above and recited in theclaims, has been made with the American Type Culture Collection (ATCC),10801 University Blvd., Manassas, Va. 20110-2209, and assigned ATCCAccession No. PTA-126155, ATCC Accession No. PTA-126156, ATCC AccessionNo. PTA-126157, and ATCC Accession No. PTA-126158, respectively. Thedate of deposit for lettuce lines SVLD9011, SVLD9012, SVLD9013, andSVLD9014 was Sep. 18, 2019. Upon issuance of a patent, all restrictionsupon the deposits will be removed, and the deposits are intended to meetall of the requirements of 37 C.F.R. § 1.801-1.809. The deposits will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced if necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

What is claimed:
 1. A lettuce plant of lettuce line SVLD9011, lettuceline SVLD9012, lettuce line SVLD9013, or lettuce line SVLD9014, a sampleof seed of said lines having been deposited under ATCC Accession No.PTA-126155, ATCC Accession No. PTA-126156, ATCC Accession No.PTA-126157, and ATCC Accession No. PTA-126158, respectively.
 2. Alettuce seed that produces the plant of claim
 1. 3. A plant part of theplant of claim 1, wherein the plant part comprises a cell of said plant.4. A lettuce plant having all the physiological and morphologicalcharacteristics of the plant of claim
 1. 5. A tissue culture ofregenerable cells of the plant of claim
 1. 6. A lettuce plantregenerated from the tissue culture of claim 5, wherein said plant hasall of the physiological and morphological characteristics of lettuceline SVLD9011, lettuce line SVLD9012, lettuce line SVLD9013, or lettuceline SVLD9014.
 7. A method of vegetatively propagating the plant ofclaim 1, the method comprising the steps of: (a) collecting tissuecapable of being propagated from the plant of claim 1; and (b)propagating a lettuce plant from said tissue.
 8. A method of introducinga trait into a lettuce plant, the method comprising: (a) utilizing as arecurrent parent the plant of claim 1 by crossing the plant with a donorlettuce plant that comprises a trait to produce F₁ progeny; (b)selecting an F₁ progeny that comprises the trait; (c) backcrossing theselected F₁ progeny with a plant of the same lettuce line used as therecurrent parent in step (a) to produce backcross progeny; (d) selectinga backcross progeny comprising the trait and the morphological andphysiological characteristics of the recurrent parent lettuce line usedin step (a); and (e) repeating steps (c) and (d) three or more times toproduce a selected fourth or higher backcross progeny.
 9. A lettuceplant produced by the method of claim
 8. 10. A method of producing alettuce plant comprising an added trait, the method comprisingintroducing a transgene conferring the trait into the plant of claim 1.11. A lettuce plant produced by the method of claim
 10. 12. A lettuceplant of lettuce line SVLD9011, lettuce line SVLD9012, lettuce lineSVLD9013, or lettuce line SVLD9014, a sample of seed of said lineshaving been deposited under ATCC Accession No. PTA-126155, ATCCAccession No. PTA-126156, ATCC Accession No. PTA-126157, and ATCCAccession No. PTA-126158, respectively, further comprising a transgene.13. The plant of claim 12, wherein the transgene confers a traitselected from the group consisting of male sterility, herbicidetolerance, insect resistance, pest resistance, disease resistance,modified fatty acid metabolism, environmental stress tolerance, modifiedcarbohydrate metabolism, and modified protein metabolism.
 14. A lettuceplant of lettuce line SVLD9011, lettuce line SVLD9012, lettuce lineSVLD9013, or lettuce line SVLD9014, a sample of seed of said lineshaving been deposited under ATCC Accession No. PTA-126155, ATCCAccession No. PTA-126156, ATCC Accession No. PTA-126157, and ATCCAccession No. PTA-126158, respectively, further comprising a singlelocus conversion.
 15. The plant of claim 14, wherein the single locusconversion confers a trait selected from the group consisting of malesterility, herbicide tolerance, insect resistance, pest resistance,disease resistance, modified fatty acid metabolism, environmental stresstolerance, modified carbohydrate metabolism, and modified proteinmetabolism.
 16. A method for producing a seed of a lettuce plant derivedfrom lettuce line SVLD9011, lettuce line SVLD9012, lettuce lineSVLD9013, or lettuce line SVLD9014, the method comprising the steps of:(a) crossing the plant of claim 1 with itself or a second lettuce plant;and (b) allowing a seed of a line SVLD9011-, line SVLD9012-, lineSVLD9013-, or line SVLD9014-derived lettuce plant to form.
 17. A methodof producing a seed of a line SVLD9011-, line SVLD9012-, line SVLD9013-,or line SVLD9014-derived lettuce plant, the method comprising the stepsof: (a) producing a line SVLD9011-, line SVLD9012-, line SVLD9013-, orline SVLD9014-derived lettuce plant from a seed produced by crossing theplant of claim 1 with itself or a second lettuce plant; and (b) crossingthe line SVLD9011-, line SVLD9012-, line SVLD9013-, or lineSVLD9014-derived lettuce plant with itself or a different lettuce plantto obtain a seed of a further line SVLD9011-, line SVLD9012-, lineSVLD9013-, or line SVLD9014-derived lettuce plant.
 18. The method ofclaim 17, the method further comprising repeating said producing andcrossing steps of (a) and (b) using the seed from said step (b) forproducing a plant according to step (a) for at least one generation toproduce a seed of an additional line SVLD9011-, line SVLD9012-, lineSVLD9013-, or line SVLD9014-derived lettuce plant.
 19. A method ofproducing food, the method comprising: (a) obtaining the plant of claim1, wherein the plant has been cultivated to maturity, and (b) collectingleaf tissue from the plant, wherein the leaf tissue is capable of use asfood.